Hematopoietic stem cell (HSC) transplants are a life-saving treatment performed on over 50,000 patients per year. Due to the high risk of failure or lethal complication, this procedure is performed only on patients with life-threatening conditions such as major hematological disorders or blood stem cell failure due to chemotherapy and radiation. Small molecules that enhance this procedure have the potential to increase the success rate and extend this treatment to a wider patient pool. HSC transplant relies on the innate abilities of hematopoietic stem cells to home to and engraft their niche in the bone marrow, so a better understanding of the cell interactions and signaling mechanisms in these niche environments could illuminate future therapeutic approaches. We conducted a chemical screen in adult zebrafish and found that 11,12 epoxyeicosatrienoic acid (EET) enhances the engraftment of transplanted hematopoietic stem cells. EETs are physiologically abundant cytochrome P450 epoxygenase derivatives of arachidonic acid with known anti-inflammatory and pro-angiogenic roles. As such, EET is a potential therapeutic to enhance HSC transplant, and has the potential to reveal new signaling pathways and cell types regulating HSC engraftment. This proposal aims to understand the mechanisms of EET signaling. In the first aim, I will work to identify the as yet unknown G-protein coupled receptor that directly binds EET. I have bioinformatically identified a list of 18 candidate receptors, and will genetically tes the requirement and sufficiency of these candidates for EET signaling in human cell lines. In the second aim I will generate stable loss of function zebrafish lines to investigate downstream mechanisms of EET signaling. These studies will focus on the known signaling mechanism of EET, whereby binding to the receptor activates Ga12/13 signaling, resulting in PI3 kinase activation and upregulation of AP-1 signaling factors. I will verify the importance of Ga12/13 subunits for this activity by creating zebrafish Ga12/13 knockout lines using CRISPR/Cas technology. Finally I will probe the cell autonomy of EET signaling in zebrafish by developing dominant negative AP-1 transgenic lines with tissue specific expression in hematopoietic stem cells and endothelial cells. Better mechanistic understanding of EET signaling will further our understanding of lipid pathways and cell-cell communication in HSC transplant, and could improve clinical bone marrow transplantation for patients with inherited blood diseases and leukemia.